Reversal of the inhibitory effects of HIV-gp120 on CD4+ T cells by stimulation through the CD28 pathway

The effects of exposure to HIV-gp120 on proliferation and cytokine production by T cell lines were investigated. T cell lines were generated by stimulation of peripheral blood mononuclear cells from several healthy donors with cross-linked anti-CD3 antibodies and IL-2. These T cell lines exhibited the characteristics of Th1 cells, producing IL-2 and interferon-gamma (IFN-γ), but not IL-4, on stimulation with anti-CD3 antibodies. In the presence of gp120, stimulation with anti-CD3 antibodies was inhibited in terms of both proliferative responses and the secretion of IL-2 and IFN-γ. Similar effects were observed when a T cell line was stimulated in the presence of a synthetic peptide representing the CD4-binding region of gp120. Neither gp120 nor the CD4-binding region peptide had any effect on IL-4 production by the T cell lines. Stimulation through the CD28 pathway partially restored both the proliferative effect and cytokine production by T cell lines in response to anti-CD3 antibodies in the presence of gp120. Anti-CD28 antibodies also partially restored cytokine production when purified CD4⁺ T cells from a T cell line were stimulated with anti-CD3 antibodies in the presence of gp120. Anti-gp120 antibodies partially or completely reversed the inhibitory effects of gp120 on T cell proliferation. These results indicate that stimulation through the CD28 pathway may restore defective CD4⁺ T cell responses in HIV-infected individuals.     

Effect of thymosin alpha-1 on subpopulations of Th1, Th2, Th17, and regulatory T cells (Tregs) in vitro

Thymosin alpha 1 (Tα1) has been shown to have beneficial effects on numerous immune system parameters, but little is known about the effects of Tα1 on patients with gastric carcinoma. The objective of this study was to determine the effect of Tα1 on subpopulations of Th1, Th2, Th17, and regulatory T cells (Tregs) in vitro, and to evaluate its efficacy as an immunoregulatory factor in patients with gastric carcinoma. We compared the effect of Tα1 on the frequency of CD4⁺ and CD8⁺ T cells, especially the CD4⁺CD25⁺Foxp3⁺ Tregs in peripheral blood mononuclear cells (PBMCs) from gastric carcinoma patients (N = 35) and healthy donors (N = 22). We also analyzed the changes in the proliferation of PBMCs in response to treatment with Tα1, and examined the production of Th1, Th2, and Th17 cytokines by PBMCs and tumor-infiltrating lymphocytes. The treatment of PBMCs from gastric cancer patients, with Tα1 (50 µg/mL) alone increased the percentage of CD4+CD25+Foxp3+ (suppressive antitumor-specific Tregs) from 1.68 ± 0.697 to 2.19 ± 0.795% (P < 0.05). Our results indicate that Tα1 increases the percentage of Tregs and IL-1β, TNF-α, and IL-6 in vitro.     

Immunological dysfunction in HIV-1-infected individuals caused by impairment of adenosine deaminase-induced costimulation of T-cell activation

The cell surface association between CD26 and adenosine deaminase (ADA) has a costimulatory function during T-cell activation. Several studies have revealed correlations among CD4⁺ CD26⁺ T-cell depletion, increased serum levels of ADA, and the evolution of human immunodeficiency virus (HIV) infection, implicating CD26 and ADA in HIV disease progression. In this context, we aimed to determine whether ADA costimulation could be altered during HIV infection. ADA costimulation was investigated in cells from HIV-infected patients (n = 36) in terms of proliferation and cytokine secretion. An effect of ADA on T-cell proliferation was found in HIV-1-infected patients and correlated positively with the CD4⁺ percentage and the nadir CD4 count and negatively with viral load, demonstrating that the response depends on the immunological status of the patient. The robust ADA-induced increase in cytokine production [interferon (IFN)-γ, interleukin (IL)-6 and IL-10] was markedly reduced in T cells from HIV-1-infected subjects. To eliminate some of the variables associated with immunological defects in HIV-1-infected patients, anti-CD3 plus ADA assays with T cells from healthy volunteers were performed in the presence of recombinant glycoprotein 120 (gp120). It was found that gp120 was responsible for the impairment of the ADA–CD26 interaction and consequently of the ADA-induced effect on both costimulation and cytokine production. The gp120-mediated disruption of the CD26–ADA interaction is a novel mechanism that might explain, at least in part, the altered immunological features observed in HIV-1-infected patients and may have significant relevance in AIDS pathogenesis.     

Mycobacterium bovis BCG Vaccination Induces Divergent Proinflammatory or Regulatory T Cell Responses in Adults

Mycobacterium bovis bacillus Calmette-Guérin (BCG), the only currently available vaccine against tuberculosis, induces variable protection in adults. Immune correlates of protection are lacking, and analyses on cytokine-producing T cell subsets in protected versus unprotected cohorts have yielded inconsistent results. We studied the primary T cell response, both proinflammatory and regulatory T cell responses, induced by BCG vaccination in adults. Twelve healthy adult volunteers who were tuberculin skin test (TST) negative, QuantiFERON test (QFT) negative, and BCG naive were vaccinated with BCG and followed up prospectively. BCG vaccination induced an unexpectedly dichotomous immune response in this small, BCG-naive, young-adult cohort: BCG vaccination induced either gamma interferon-positive (IFN-γ⁺) interleukin 2-positive (IL-2⁺) tumor necrosis factor α-positive (TNF-α⁺) polyfunctional CD4⁺ T cells concurrent with CD4⁺ IL-17A⁺ and CD8⁺ IFN-γ⁺ T cells or, in contrast, virtually absent cytokine responses with induction of CD8⁺ regulatory T cells. Significant induction of polyfunctional CD4⁺ IFN-γ⁺ IL-2⁺ TNF-α⁺ T cells and IFN-γ production by peripheral blood mononuclear cells (PBMCs) was confined to individuals with strong immunization-induced local skin inflammation and increased serum C-reactive protein (CRP). Conversely, in individuals with mild inflammation, regulatory-like CD8⁺ T cells were uniquely induced. Thus, BCG vaccination either induced a broad proinflammatory T cell response with local inflammatory reactogenicity or, in contrast, a predominant CD8⁺ regulatory T cell response with mild local inflammation, poor cytokine induction, and absent polyfunctional CD4⁺ T cells. Further detailed fine mapping of the heterogeneous host response to BCG vaccination using classical and nonclassical immune markers will enhance our understanding of the mechanisms and determinants that underlie the induction of apparently opposite immune responses and how these impact the ability of BCG to induce protective immunity to TB.     

Association of T cell dysfunction with the presence of IgG autoantibodies on CD4+ lymphocytes in haemophilia patients; results of a 10-year study

HIV induces progressive dysfunction followed by numerical depletion of CD4⁺ lymphocytes. IgG autoantibodies and gp120-containing immune complexes have been implicated in the pathogenesis of AIDS. We carried out a longitudinal study in 19 HIV^– and 72 HIV⁺ haemophilia patients over a 10-year period in order to investigate a possible relationship between the occurrence of autoantibodies and CD4⁺ lymphocyte changes. IgM, IgG, C3d and gp120 on the surface of CD4⁺ lymphocytes were determined in heparinized whole blood with flow cytometry and double-fluorescence. The in vitro response of autoantibody-coated cells was tested in cell cultures with concanavalin A (Con A), phytohaemagglutinin (PHA), pokeweed mitogen (PWM), anti-CD3 MoAb or pooled allogeneic stimulator cells (MLC). After a 10-year follow up, 12 of 71 HIV⁺ and 16 of 19 HIV^– haemophilia patients showed no evidence of immunoglobulins on circulating CD4⁺ lymphocytes. HIV^– haemophilia patients without autoantibodies had CD4⁺and CD8⁺ cell counts in the normal range (957 ± 642/μl and 636 ± 405/μl) and normal T cell responses in vitro (mean relative response (RR) ≥ 0.7). In contrast, HIV⁺ haemophilia patients showed immunological abnormalities which were associated with the autoantibody and immune complex load of CD4⁺ blood lymphocytes. HIV⁺ patients without autoantibodies had a mean CD4⁺ lymphocyte count of 372 ± 274/μl, a mean CD8⁺ lymphocyte count of 737 ± 435/μl, and normal T lymphocyte stimulation in vitro (mean RR ≥ 0.7). HIV⁺ patients with complement-fixing IgM on CD4⁺ lymphocytes had somewhat lower CD4⁺ (255 ± 246/μl, P= NS) and CD8⁺ (706 ± 468/μl, P= NS) lymphocyte numbers, and also normal T lymphocyte stimulation (mean RR ≥ 0.7) in vitro. However, patients with complement-fixing IgG autoantibodies showed a strong decrease of CD4⁺ (150 ± 146/μl, P< 0.02) and CD8⁺ (360 ± 300/μl, P< 0.02) lymphocytes and impaired CD4⁺ lymphocyte stimulation in vitro with a mean RR of 0.5 ± 0.5 for Con A (P= NS), 0.7 ± 0.8 for PHA (P< 0.03), 0.4 ± 0.4 for PWM (P= NS), 0.8 ± 1.2 for anti-CD3 MoAb (P< 0.04) and 0.7 ± 1.0 for pooled allogeneic stimulator cells (P= 0.05). Patients with gp120-containing immune complexes on CD4⁺ blood lymphocytes demonstrated strongly decreased CD4⁺(25 ± 35/μl, P< 0.0001) and CD8⁺ (213 ± 212/μl, P< 0.006) lymphocyte counts as well as strongly impaired T lymphocyte responses in vitro upon stimulation with PHA (RR 0.2 ± 0.1, P < 0.02), PWM (RR 0.2 ± 0.2, P= 0.05), anti-CD3 MoAb (RR 0.1 ± 0.1, P< 0.04), and allogeneic stimulator cells (RR 0.2 ± 0.1, P< 0.02). These data led us to speculate that autoantibody formation against CD4⁺ lymphocytes is an important mechanism in the pathogenesis of AIDS. We hypothesize that autoantibodies against circulating CD4⁺ lymphocytes inhibit CD4⁺ cell function, especially the release of cytokines, and induce CD4⁺ cell depletion. The reduction and dysfunction of CD4⁺ lymphocytes may be responsible for the CD8⁺ cell depletion observed in HIV⁺ patients.     

Preferential S Phase Entry and Apoptosis of CD4 T Lymphocytes of HIV-1-Infected Patients after in Vitro Cultivation

We have studied the relationship between spontaneous apoptosis and cell cycle perturbations in circulating peripheral blood lymphocytes of HIV-1-infected patients and healthy controls. PBMC obtained from HIV-1-infected patients and healthy controls were incubated in culture medium for 48 h. Cells were separated into CD4⁺ and CD8⁺ populations using immunomagnetic beads. Apoptosis and cell cycle phases were measured by propidium iodide staining and bromodeoxyuridine (BrdU) incorporation followed by flow cytometric analyses. In experiments using cells obtained from HIV-1-infected patients, spontaneous apoptosis was more frequent in CD4⁺ T lymphocytes than in CD8⁺ T lymphocytes (17.6% vs 9.5%, P < 0.005). Among healthy controls, spontaneous apoptosis in CD4⁺ and CD8⁺ T lymphocytes was comparable (4.5% vs 5.1%). Lymphocytes obtained from patients were more frequently in S phase than healthy controls' cells (2.2 ± 0.9% vs 0.5 ± 0.2%, P < 0.002) and patients' CD4⁺ cells tended to enter S phase more frequently than controls' CD4⁺ cells (4.2% ± 3.5% vs 1.8% ± 0.5% P < 0.04), whereas the frequency of S phase CD8⁺ T cells was not different among patients (2.8% ± 2.9%) and controls (1.8% ± 0.5%) (P > 0.4). Kinetic analyses using BrdU and PI staining revealed that S phase cells were more likely to become apoptotic than resting (G₀–G₁) cells (28.4% ± 10.3% vs 11.3% ± 9.9% in patients, P < 0.04, and 15.3% ± 2.8% vs 1.8% ± 0.5% in controls, P < 0.003). Lymphocytes obtained from HIV-1-infected persons are activated in vivo to enter S phase and to undergo spontaneous apoptosis after brief in vitro cultivation. The present studies indicate that most apoptotic cells in this system are CD4⁺ and kinetic analyses reveal that S phase cells are more likely to undergo spontaneous apoptosis than G₀–G₁ cells. Accelerated cell death in HIV-1 disease may contribute to the failure of lymphocyte responsiveness to appropriate T cell receptor stimulation.     

Regulatory T cell levels and cytokine production in active non-infectious uveitis: in-vitro effects of pharmacological treatment

The aim of this study was to quantify the proportion of regulatory T cells (T_reg) and cytokine expression by peripheral blood mononuclear cells (PBMCs) in patients with active non-infectious uveitis, and to evaluate the effect of in-vitro treatment with infliximab, dexamethasone and cyclosporin A on T_reg levels and cytokine production in PBMCs from uveitis patients and healthy subjects. We included a group of 21 patients with active non-infectious uveitis and 18 age-matched healthy subjects. The proportion of forkhead box protein 3 (FoxP3)⁺ T_reg cells and intracellular tumour necrosis factor (TNF)-α expression in CD4⁺ T cells was determined by flow cytometry. PBMCs were also either rested or activated with anti-CD3/anti-CD28 and cultured in the presence or absence of dexamethasone, cyclosporin A and infliximab. Supernatants of cultured PBMCs were collected and TNF-α, interleukin (IL)-10, IL-17 and interferon (IFN)-γ levels were measured by enzyme-linked immunosorbent assay (ELISA). No significant differences were observed in nT_reg levels between uveitis patients and healthy subjects. However, PBMCs from uveitis patients produced significantly higher amounts of TNF-α and lower amounts of IL-10. Dexamethasone treatment in vitro significantly reduced FoxP3⁺ T_reg levels in PBMCs from both healthy subjects and uveitis patients, and all tested drugs significantly reduced TNF-α production in PBMCs. Dexamethasone and cyclosporin A significantly reduced IL-17 and IFN-γ production in PBMCs and dexamethasone up-regulated IL-10 production in activated PBMCs from healthy subjects. Our results suggest that PBMCs from patients with uveitis express more TNF-α and less IL-10 than healthy subjects, and this is independent of FoxP3⁺ T_reg levels. Treatment with infliximab, dexamethasone and cyclosporin A in vitro modulates cytokine production, but does not increase the proportion of FoxP3⁺ T_reg cells.     

Generalized immune activation in pulmonary tuberculosis: co-activation with HIV infection

Parameters of immune activation/differentiation were studied in a group of newly diagnosed HIV⁻ and HIV⁺ pulmonary tuberculosis (TB) patients. Compared with controls, HLA-DR expression on both CD4 and CD8 T cells from the HIV⁻ TB patients was approximately doubled; HLA-DR on T cells from the HIV⁺ group was tripled. The monocytes from both groups of patients expressed abnormally high levels of the Fcγ receptors I and III. Serum levels of tumour necrosis factor-alpha (TNF-α), neopterin and β₂-microglobulin were increased in HIV⁻ and even more so in HIV⁺ TB patients. The expression of HLA-DR on T cell subsets and of FcγR on monocytes correlated with each other, but not with serum activation markers. This pattern of non-specific activation during TB infection may be associated with enhanced susceptibility to HIV infection.     

Administration of anti-CD3 monoclonal antibody during experimental Chagas’ disease induces CD8+ cell-dependent lethal shock

The injection of the 145-2C11 anti-CD3 MoAb in mice induces a polyclonal T cell activation resulting in the release of several cytokines, including interferon-gamma (IFN-γ) and tumour necrosis factor-alpha (TNF-α). As these cytokines are known to be involved in the host defence against Trypanosoma cruzi, we measured serum levels of IFN-γ and TNF-α after injection of the 145-2C11 MoAb in the course of experimental murine Chagas'' disease. Compared with control mice, T. cruzi-infected BALB/c mice were found to be primed to secrete very high levels of IFN-γ and TNF-α from the second and the first week of infection, respectively, up to the chronic phase. In vivo cell depletion experiments indicated that CD8⁺ T cells were responsible for these dramatic hyperproductions of IFN-γ and TNF-α. While all control mice survived anti-CD3 MoAb injection, a high lethality rate was observed in T. cruzi-infected mice within 24 h after anti-CD3 MoAb challenge. Pretreatment with neutralizing anti-IFN-γ MoAb or depletion of CD8⁺ T cell population dramatically decreased the mortality induced by anti-CD3 MoAb in T. cruzi-infected mice. Finally, we showed that anti-CD3 MoAb injection in T. cruzi-infected mice was followed by a massive release of nitric oxide (NO) metabolites, which was partially reduced by IFN-γ or TNF-α neutralization. The administration of the NO synthase inhibitor n-nitro-l-arginine methyl ester (L-NAME) before anti-CD3 MoAb challenge did not prevent and even enhanced lethality in T. cruzi-infected mice, suggesting that NO overproduction and lethal shock are not causally related. We conclude that injection of anti-CD3 MoAb in the course of experimental Chagas’ disease induces a CD8⁺ cell-dependent shock mediated by IFN-γ and TNF-α.     

In Vitro Exposure to Highly Cytopathic HIV-1 X4 Strains Increases Expression of Mucosa-Associated Integrins on CD4 T Cells

To determine whether infection with HIV-1 strains of different tropisms would influence expression of the mucosa-associated integrins α4β7 and αEβ7 or the lymph node homing receptor L-selectin on peripheral T lymphocytes, cells were infected with the CXCR4-tropic (X4)/syncytium-inducing (SI) HIV-1_IIIB strain or with X4/SI or CCR5-tropic (R5)/non-SI (NSI) primary human isolates. Flow cytometric analyses of CD4⁺ T cells from cultures infected with HIV-1_IIIB and one X4/SI primary HIV-1 isolate revealed a significant increase in surface expression of α4β7 and αEβ7 12 days after infection. L-selectin expression was not significantly affected on CD4⁺ T cells. However, infection with another X4/SI and two R5/NSI primary HIV-1 isolates did not significantly alter homing receptor expression on CD4⁺ T cells. Since a higher degree of CD4 cytopathicity occurred in those cultures having increased integrin expression, these data suggest that significantly altered mucosal homing receptor expression on CD4⁺ T cells may result as a “bystander” effect after infection with some cytopathic isolates of HIV-1.     

Intracellular cytokine production by human CD4+ and CD8+ T cells from normal and immunodeficient donors using directly conjugated anti-cytokine antibodies and three-colour flow cytometry

Using three-colour flow cytometry, we have measured intracellular IL-2, interferon-gamma (IFN-γ) and tumour necrosis factor-alpha (TNF-α) induced in human CD4⁺ and CD8⁺ T cells from normal donors and patients with common variable immunodeficiency (CVID). Since a new range of directly FITC-conjugated anti-cytokine antibodies was used, conditions were optimized for the concentration of antibody, for cell permeabilization and fixation, and for the time of exposure to monensin to retain the cytokines within the cell. Kinetics of intracellular cytokine production were measured for up to 20h in culture with phorbol myristate acetate (PMA) and ionomycin, or with phytohaemagglutinin (PHA). Kinetic studies of activation with PMA and ionomycin show that a higher proportion of normal CD4⁺ cells can make IL-2 than the other two cytokines, and that there are more TNF-α-positive CD4⁺ cells than cells with IFN-γ. For normal CD8⁺ cells the highest production of cytokine is of IFN-γ (up to 50% of the cells) especially at longer times (10–20h) of stimulation. For CD8⁺ cells, IL-2-positive cells exceed those with TNF-α. The other mitogenic stimulus used (PHA) was grossly inferior to PMA and ionomycin in its ability to induce intracellular cytokines. The time of exposure to monensin was also examined. Its continuous presence in the cultures (up to a maximum of 20h) increased the detection of IL-2-positive cells without apparently reducing the percentage of cytokine-positive CD4⁺ or CD8⁺ cells. Finally, using optimal conditions, we compared cytokine production in cells from patients with the disease CVID and showed normal cellular levels of ability to produce IL-2 and TNF-α but significantly raised levels of production of IFN-γ in both CD4⁺ and CD8⁺ lymphocytes. This suggests that the pathology of this disease may involve an excessive Th1-type response.     

The enigma of CD57+CD28– T cell expansion—anergy or activation?

Expansion of a CD57⁺CD8^– T lymphocyte subset has been reported in HIV and human cytomegalovirus (HCMV) infection. Almost all of these T cells lack CD28 expression. While CD28^– cells are often associated with anergy, some authors believe their expansion in HIV infection precipitates immunodeficiency. We studied 15 randomly chosen patients with immune activation and observed that CD57⁺CD28^– T cell expansion may occur in various conditions and to the same degree as in HIV infection without resulting in immunodeficiency. Triple colour flow cytometry also revealed that the CD57 and CD28 antigens are coexpressed in only 3% of CD8⁺ T cells, irrespective of the underlying condition, so that almost all CD57⁺CD8⁺ cells are always CD28^–. Analysis of Fas (CD95) expression with respect to CD28 expression on CD4⁺ and CD8⁺ T cells from 10 additional patients indicated no increased commitment to apoptosis in CD28^– T cells. Semiquantitative polymerase chain reaction (PCR) comparing CD28⁺ and CD28^–CD8⁺ T cells with respect to cytokine gene expression (tumour necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), IL-1β) in five renal transplant patients with expansion of the CD57⁺ subset detected no cytokine gene expression deficit in CD28^– T cells. A direct association of increased proportions of CD57⁺CD28^–CD8⁺ T cells with immunodeficiency/anergy is disputed.     

Cytolytic mechanisms and T-cell receptor Vβ usage by ex vivo generated Epstein–Barr virus-specific cytotoxic T lymphocytes

Ex-vivo-generated Epstein–Barr virus (EBV)-specific cytotoxic T lymphocytes (CTL) have been used for cellular adoptive immunotherapy of EBV-associated lymphomas. Here we investigated the phenotypes, cytolytic mechanisms, polyfunctionality and T-cell receptor (TCR) usage in growing and established CTL, generated by weekly stimulation with an EBV-transformed autologous lymphoblastoid cell line (LCL). Our results showed that phenotypically mature CTL developed within the first 4 weeks of culture, with an increase in CD45RO and CD69, and a decrease in CD45RA, CD62L, CD27 and CD28 expression. Spectratyping analysis of the variable β-chain of the TCR revealed that TCR repertoire remained diverse during the course of culture. Cytotoxicity of CTL was significantly inhibited by concanamycin A (P < 0·0001) and ethylene glycol-bis tetraacetic acid (P < 0·0001), indicating that a calcium and perforin-mediated exocytosis pathway with the release of granzyme B was the principal cytotoxic mechanism. The CTL mainly produced interferon-γ (IFN-γ) or tumour necrosis factor-α (TNF-α) upon restimulation with autologous LCL, although there were some polyfunctional cells producing IFN-γ and TNF-α. Granzyme B, perforin and Fas ligand were detected in CD8⁺ and CD4⁺ cells in all CTL; however, a greater proportion of CD8⁺ than CD4⁺ T cells expressed granzyme B (P < 0·0001) and more granzyme B was detected in CD8⁺ T cells than in CD4⁺ T cells (P = 0·001). This difference was not observed with Fas ligand or perforin expression. Our results provide insight into the basic characteristics of ex-vivo-generated CTL.     

Infants with late breast milk acquisition of HIV-1 generate interferon-gamma responses more rapidly than infants with early peripartum acquisition

Infants infected with HIV-1 after the first month of life have a lower viral set-point and slower disease progression than infants infected before 1 month. We investigated the kinetics of HIV-1-specific CD8⁺ T lymphocyte secretion of interferon (IFN)-γ in infants infected before 1 month of life compared with those infected between months 1 and 12 (late infection). HIV-1 infection was assessed at birth and at months 1, 3, 6, 9 and 12 and timing of infection was determined by HIV-1 gag DNA from dried blood spots and verified by plasma HIV-1 RNA levels. HIV-1 peptide-specific IFN-γ responses were measured by enzyme-linked immunospot at months 1, 3, 6, 9 and 12. Timing of development of IFN-γ responses was compared using the log–rank test and Kaplan–Meier survival curves. Infants infected late developed HIV-1-specific CD8⁺ T cell responses 2·8 months sooner than infants infected peripartum: 2·3 versus 5·1 months after HIV-1 infection (n = 52, P = 0·04). Late-infected infants had more focused epitope recognition than early-infected infants (median 1 versus 2 peptides, P = 0·03); however, there were no differences in the strength of IFN-γ responses. In infants infected with HIV-1 after the first month of life, emergence of HIV-1-specific CD8⁺ IFN-γ responses is coincident with the decline in viral load, nearly identical to what is observed in adults and more rapid than in early-infected infants.     

Measurement of Phenotype and Absolute Number of Circulating Heparin-Binding Hemagglutinin,ESAT-6 and CFP-10, and Purified Protein Derivative Antigen-Specific CD4 T Cells Can Discriminate Active from Latent Tuberculosis Infection

The tuberculin skin test (TST) and interferon gamma (IFN-γ) release assays (IGRAs) are used as adjunctive tests for the evaluation of suspected cases of active tuberculosis (TB). However, a positive test does not differentiate latent from active TB. We investigated whether flow cytometric measurement of novel combinations of intracellular cytokines and surface makers on CD4 T cells could differentiate between active and latent TB after stimulation with Mycobacterium tuberculosis-specific proteins. Blood samples from 60 patients referred to the Singapore Tuberculosis Control Unit for evaluation for active TB or as TB contacts were stimulated with purified protein derivative (PPD), ESAT-6 and CFP-10, or heparin-binding hemagglutinin (HBHA). The CD4 T cell cytokine response (IFN-γ, interleukin-2 [IL-2], interleukin-17A [IL-17A], interleukin-22 [IL-22], granulocyte-macrophage colony-stimulating factor [GM-CSF], and tumor necrosis factor alpha [TNF-α]) and surface marker expression (CD27, CXCR3, and CD154) were then measured. We found that the proportion of PPD-specific CD4 T cells, defined as CD154⁺ TNF-α⁺ cells that were negative for CD27 and positive for GM-CSF, gave the strongest discrimination between subjects with latent and those with active TB (area under the receiver operator characteristic [ROC] curve of 0.9277; P < 0.0001). Also, the proportions and absolute numbers of HBHA-specific CD4 T cells were significantly higher in those with latent TB infection, particularly CD154⁺ TNF-α⁺ IFN-γ⁺ IL-2⁺ and CD154⁺ TNF-α⁺ CXCR3⁺. Finally, we found that the ratio of ESAT-6- and CFP-10-responding to HBHA-responding CD4 T cells was significantly different between the two study populations. In conclusion, we found novel markers of M. tuberculosis-specific CD4 cells which differentiate between active and latent TB.     

Depletion of γδ+ T cells increases CD4+ FoxP3 (T regulatory) cell response in coxsackievirus B3-induced myocarditis

Coxsackievirus B3 (CVB3) causes severe myocarditis in BALB/c mice which depends upon CD4⁺ T helper type 1 [Th1; i.e. interferon-γ⁺ (IFN-γ⁺)] and γδ⁺ cells. Depleting γδ⁺ cells using anti-γδ antibody suppresses myocarditis and CD4⁺ IFN-γ⁺ cell numbers in the spleen and heart of infected mice while increasing CD4⁺ FoxP3⁺ cells. Mice deficient in γδ⁺ cells have increased numbers of naïve (CD44^lo CD62L^hi) and fewer effector (CD44^hi CD62^lo) memory CD4⁺ cells than infected γδ⁺-cell-sufficient mice. Virus neutralizing antibody titres are not significantly different between γδ⁺ T-cell-sufficient and -deficient animals. To confirm that the memory cell response differs in acutely infected mice lacking γδ⁺ cells, CD4⁺ cells were purified and adoptively transferred into naïve recipients, which were rested for 4 weeks then infected with CVB3. Recipients given either 0·5 × 10⁶ or 1·0 × 10⁶ CD4⁺ from infected donors developed over twice the severity myocarditis and 10-fold less cardiac virus titre compared with recipients given equivalent numbers of CD4⁺ cells from infected and γδ⁺-cell-depleted donor animals. Additionally, to show that more functionally active T regulatory cells are present in γδ⁺ T-cell-depleted mice, CD4⁺ CD25⁺ and CD4⁺ CD25⁻ cells were isolated and adoptively transferred into infected recipients. Mice receiving CD4⁺ CD25⁺ cells from γδ⁺ T-cell-depleted donors developed significantly less myocarditis and CD4⁺ Th1 cell responses compared with mice receiving equal numbers of CD4⁺ CD25⁺ cells from infected γδ⁺ T-cell-sufficient animals. This study shows that γδ⁺ cells promote CD4⁺ IFN-γ⁺ acute and memory responses by limiting FoxP3⁺ T regulatory cell activation.     

Assessment of the Phenotype and Functionality of Porcine CD8 T Cell Responses following Vaccination with Live Attenuated Classical Swine Fever Virus (CSFV) and Virulent CSFV Challenge

Vaccination with live attenuated classical swine fever virus (CSFV) induces solid protection after only 5 days, which has been associated with virus-specific T cell gamma interferon (IFN-γ) responses. In this study, we employed flow cytometry to characterize T cell responses following vaccination and subsequent challenge infections with virulent CSFV. The CD3⁺ CD4⁻ CD8^hi T cell population was the first and major source of CSFV-specific IFN-γ. A proportion of these cells showed evidence for cytotoxicity, as evidenced by CD107a mobilization, and coexpressed tumor necrosis factor alpha (TNF-α). To assess the durability and recall of these responses, a second experiment was conducted where vaccinated animals were challenged with virulent CSFV after 5 days and again after a further 28 days. While virus-specific CD4 T cell (CD3⁺ CD4⁺ CD8α⁺) responses were detected, the dominant response was again from the CD8 T cell population, with the highest numbers of these cells being detected 14 and 7 days after the primary and secondary challenges, respectively. These CD8 T cells were further characterized as CD44^hi CD62L⁻ and expressed variable levels of CD25 and CD27, indicative of a mixed effector and effector memory phenotype. The majority of virus-specific IFN-γ⁺ CD8 T cells isolated at the peaks of the response after each challenge displayed CD107a on their surface, and subpopulations that coexpressed TNF-α and interleukin 2 (IL-2) were identified. While it is hoped that these data will aid the rational design and/or evaluation of next-generation marker CSFV vaccines, the novel flow cytometric panels developed should also be of value in the study of porcine T cell responses to other pathogens/vaccines.     

Integration of B cells and CD8+ T in the protective regulation of systemic epithelial inflammation

Mechanisms that control abnormal CD4⁺ T cell-mediated tissue damage are a significant factor in averting and resolving chronic inflammatory epithelial diseases. B cells can promote such immunoregulation, and this is thought to involve interaction with MHC II- or CD1-restricted regulatory T cells. The purpose of this study is to genetically define the interacting cells targeted by protective B cells, and to elucidate their regulatory mechanisms in CD4⁺ T cell inflammation. Transfer of Gαi2−/− CD3⁺ T cells into lymphopenic mice causes a dose-dependent multi-organ inflammatory disease including the skin, intestine, and lungs. Disease activity is associated with elevated levels of serum TNF-α and IFN-γ, and an activated IL-17 producing CD4⁺ T cell population. Mesenteric node B cells from wild type mice suppress disease activity, serum cytokine expression, and levels of CD4⁺ T cells producing TNF-α IFN-γ, and IL-17. The protective function of B cells requires genetic sufficiency of IL-10, MHC I and TAP1. Regulatory B cells induce the expansion and activation of CD8⁺ T cells, which is correlated with disease protection. These results demonstrate that CD8⁺ T cells can ameliorate lymphopenic systemic inflammatory disease, through peptide/MHC I-dependent B cell interaction.     

β2 Integrin deficiency yields unconventional double-negative T cells distinct from mature classical natural killer T cells in mice

Expressed on leucocytes, β₂ integrins (CD11/CD18) are specifically involved in leucocyte function. Using a CD18-deficient (CD18^−/−) mouse model, we here report on their physiological role in lymphocyte differentiation and trafficking. CD18^−/− mice present with a defect in the distribution of lymphocytes with highly reduced numbers of naïve B and T lymphocytes in inguinal and axillary lymph nodes. In contrast, cervical lymph nodes were fourfold enlarged harbouring unconventional T-cell receptor-αβ (TCR-αβ) and TCR-γδ CD3⁺ CD4⁻ CD8⁻ (double-negative; DN) T cells that expanded in situ. Using adoptive transfer experiments, we found that these cells did not home to peripheral lymph nodes of CD18^wt recipients but, like antigen-experienced T or natural killer (NK) T cells, recirculated through non-lymphoid organs. Lacking regulatory functions in vitro, CD18^−/− TCR-αβ DN T cells did not suppress the proliferation of polyclonally activated CD4⁺ or CD8⁺ (single-positive; SP) T cells. Most interestingly, CD18^−/− TCR-αβ DN T cells showed intermediate TCR expression levels, an absent activation through allogeneic major histocompatibility complex and a strong proliferative dependence on interleukin-2, hence, closely resembling NKT cells. However, our data oppose former reports, clearly showing that, because of an absent reactivity with CD1d-αGalCer dimers, these cells are not mature classical NKT cells. Our data indicate that CD18^−/− TCR-αβ DN T cells, like NKT and TCR-γδ T cells, share characteristics of both adaptive and innate immune cells, and may accumulate as a compensatory mechanism to the functional defect of adaptive immunity in CD18^−/− mice.